3-Methylthiorifamycins

ABSTRACT

A new species of Micromonospora, designated Micromonospora lacustris sp. nov. Routien, when subjected to submerged aerobic fermentation under controlled conditions, produces a mixture of at least 13 antibiotics. Two of these antibiotics are rifamycin S and rifamycin SV. Two other members of the antibiotic mixture are the 3-thiomethyl derivatives of rifamycin S and rifamycin SV.

United States Patent Celmer et al.

Oct. 21, 1975 3-METHYLTHIORIFAMYCINS Inventors: Walter D. Celmer; FrankC.

Sciavolino, both of New London; Walter P. Cullen, East Lyme; John B.Routien, Lyme, all of Conn.

Assignee: Pfizer Inc., New York, N.Y.

Filed: Jan. 9, 1974 Appl. No.: 431,845

US. Cl. 260/239.3 P; 424/117; 195/80 R Int. Cl. C07D 201/02 Field ofSearch 260/2393 1? References Cited UNITED STATES PATENTS 12/1971 Maggi260/2393 Primary Examinerl-lenry R. Jiles Assistant Examiner-Bernard lDentz Attorney, Agent, or FirmC0nn0lly and Hutz [57] ABSTRACT 4 Claims,1 Drawing Figure US. Patent Oct. 21, 1975 O Q a: w Peraerzi fiwwmiiimae3 -METI'IYLTH IORIFAMYCINS BACKGROUND OF THE INVENTION The rifamycins, agroup of closely related antibiotics, are described by Sensi et al.:Farmaco, Ed. Sci. 14, 146 (1959); Antibiotics Ann. 1959/1960, 262(1960a); Experientia 16, 412 (1960b); Farmaco, Ed. Sci. 16, 165 (1961);and Res. Progr. Org. Biol. Med. Chem. 1, 337 (1964).

The constitution of the rifamycins was established by Prelog, V., PureAppl. Chem. 7, 551 (1963b). Prelog coined the term ansamycin to refer tothis particular class of large molecule antibiotics.

SUMMARY OF THE INVENTION This invention is concerned with a mixture ofansamycins produced by a new species of Micromonospora, designatedMicromonospora Iacustris sp. nov. Routien. Two members of the ansamycinmixture are rifamycin S and rifamycin SV. Two other ansamycihs are the3- thiomethyl derivatives of rifamycin S and rifamycin SV.

DETAILED DESCRIPTION OF THE INVENTION The microorganism useful for thepreparation of the antibiotics of this invention was isolated from a mudsample from Rogers Lake, Conn. This culture (Pfizer F.D. 23849),designated Micromonospora lacustris sp. nov. Routien, has been depositedin the American Type Culture Collection, Rockville, Md., and added toits collection, representative as the type culture as ATCC 21975.

Identification media used for the characterization of M. lacustris andreferences for their composition are as follows:

1. Bennett's Agar (and 0.1% CaCO Waksman, SA. The Actinomycetes vol. 2,1961. Medium 30 on p. 331.

2. Emersons Agar (and 0.1% CaCO Waksman,

1961. Medium 28 on p.331.

3. Tomato-Paste Oatmeal Agar (and 0.1% CaCO Waksman, 1961. Medium 34 onp. 332.

4. Glucose-Asparagine Agar (and 0.1% CaCO Waksman, 1961. Medium 2 on p.328.

5. Glucose-Yeast Extract Agar (and 0.1% CaCO M.J. Weinstein et. a1.Antimicrobial Agents and Chemotherapy p. 436, 1968.

6. Starch a. Waksman, 1961. Medium 21 on p. 330.

b. As above but with 1% yeast extract added.

c. Gordon and Mihm, J. Bact. 73, -27, 1957.

Potato starch 20.0 g. Ammonium chloride 0.5 g. Agar 15.0 g.

Distilled water 1.0 liter 7. Gelatin Waksman, 1961. Medium on p. 330.

8. Tyrosine Waksman, 1961. Medium 11 on p. 329.

9. Czapek-Sucrose Waksman, 1961. Medium 1 on p.

10. Potato Slice Luedemann, GM. and Brodsky, B.,

Antimicrobial Agents and Chemotherapy p. 47-52, 1965.

1 1. Potato Slice plus CaCO Luedmann and Brodsky,

12. Carrot Plugs.

l3. Tap Water Agar (2%).

14. Peptone Iron Agar Waksman, 1961. Medium 38 on p. 332.

15. Difco Skim Milk.

l6. ATCC Medium 172 ATCC Catalog of Strains, 9th

Edition, p. 172, 1970.

17. Dextrose Nitrate Broth Waksman, 1961. Medium 18. Organic NitrateBroth Waksman, 1961. Medium 37 on p. 332.

19. Sucrose Invertase Levine, M. and Schoenlein, H.W., A Compilation ofCulture Media, 1930. Medium 622 on p. 176.

20. Cellulose Levine and Schoenlein, 1930. Medium 2511 on p. 823.

21. Cellulose Jensen, I-I.L., Proc. Linnean Soc. N.S.

Wales 55, 231 (1930).

22. Nitrogen Utilization Weinstein et al., Antimicrobial Agents andChemotherapy p. 437, 1968.

23. Carbohydrate Utilization Weinstein et a1.

M. lacustris was planted in tubes or petri dishes at least in duplicatefor some long range tests. Incubation was at 28C. except where otherwisenoted. Readings were made at various intervals up to 14 days with sometests continued for longer periods of time. The color designations referto color of chips in Color Harmony Manua 4th edition, 1958, published bythe Container Corporation of America, U.S.A., but descriptive terms arethose of the investigator. The methods are mainly those described byM.J. Weinstein et al. in Antimicrobial Agents and Chemotherapy 435-437(1967/1968). The description of M. lacustris ATCC 21975 is as follows:

Characteristic of the genus Micromonospora, M. lacustris has no aerialmycelium. These are spores only on the substrate mycelium, and these areborne singly on hyphae. Spores produced on tap water agar are rare,terminal, sessile, arranged at random in culture, mostly 1.1-1.5 micronswide, sometimes 2.0 microns wide. Infrequent intercalary swellings arefound. On glucoseyeast extract agar the spores are similar but aresometimes numerous in brownish spots in the mycelium.

Yeast extract and NZ-Amine A (Sheffield Chemical Co.) provide for goodgrowth. Sodium nitrate, asparagine and glutamic acid are not utilized.Growth is poor to good on ATCC Medium 172 at 2l, 28 and 37C. but thereis inconsistent growth at 45C.

Nitrate reduction: no reduction of nitrate to nitrite in 21 days ineither dextrose nitrate broth or organic nitrate broth.

H 5 production: (4 days, lead acetate strip test) production frompeptone iron agar.

Cellulose: no growth in either medium even after 38 days.

Gelatin: liquified.

Starch: no hydrolysis on media 60 and 6b but good hydrolysis on media 6cand 6d.

Sucrose inversion: positive.

Skimmed milk: no change at 21 days with later coagulation and partialpeptonization occurring.

Carbon utilization: utilized glucose, galactose, fructose, D-mannose,d(-)ribose, starch, sucrose, trehalose and xylose; not utilizedadonitol, L-arabinose, cellulose, dulcitol, inositol, D-mannitol,raffinose, rhammose and d(-)sorbitol; variable lactose and D- melibiose.

Additional cultural data pertaining to M. lacustris ATCC 21975 arecontained in the following table:

Color of Soluble Medium Growth mycelium pigment Bennct's agar CaCO Good,flat with Bright orange Bright yellow some roughening (near 4 pa)Emerson's agar CaCO Good. raised, Bright orange Brown roughened.wrinkled (5 pa to 5 pc) Tomato paste oatmeal CaCO Good to excellent,Orange (4 pa to Slight brown flat, smooth 5 la) Glucose-yeast extractCaCO Moderate to good, Orange (4 na to Brown flat or raised and 5 pa)roughened Glucose asparagine agar plus Essentially no growth CaCo Czapeksucrose agar Essentially no growth Tyrosine agar Essentially no growthPotato slice Good. roughened or Reddish orange None thin, flat (5 pc to6 pa) Potato slice CaCO; Good, wrinkled Reddish orange None (near 5 pc)Gelatin Poor to moderate, Pale yellowish None flat, thin orange (near 3pc) Carrot plugs No growth Starch agar Essentially no growth on media 6aand 6b but good growth on 6e and 6d A mutant strain (Pfizer F.D. 24189),developed by mutagenic treatment of M. lacustris ATCC 21975, wasdeposited with the American Type Culture Collection and designated ATCC21974. The mutant strain has a duller color than the parent culture onglucose-yeast agar plus CaCO and other media. The mutant strain producesmore spores and slightly larger spores than the parent culture. Themutant strain, in contrast to the parent culture, cannot utilizefructose and lactose as carbon sources. The mutant strain produceslesser amounts of rifamycin S and rifamycin SV in the employedfermentation media than does the parent culture.

Cultivation of M. lacustris preferably takes place in aqueous nutrientmedia at a temperature of about 24-36C., and under aerobic, submergedconditions with agitation. Nutrient media which are useful for suchpurposes include a source of assimilable carbon such as sugars, starch,glycerol and molasses; a source of organic nitrogen such as casein,enzymatic digest of casein, meat meal, wheat gluten, cotton seed meal,soybean meal and peanut meal. A source of growth substances such asdistillers solubles and/or yeast extract as well as salts such as sodiumchloride, ammonium acetate, ammonium sulfate, potassium phosphate andtrace minerals such as iron, magnesium, zinc, cobalt and manganese mayalso be utilized with advantageous results. If excessive foaming isencountered during fermentation, antifoam agents such as vegetable oilsor silicones may be added to the fermentation medium. The pH of thefermentation tends to remain rather constant but if variations areencountered, a buffering agent such as calcium carbonate may also beadded to the medium. Aeration of the medium in tanks for submergedgrowth is preferably maintained at the rate of about /2 to 2 volumes offree air per volume of broth per minute. Agitation may be maintained bymeans of agitators generally familiar to those in the fermentationindustry. Aseptic conditions must, of course, be maintained through thetransfer of the microorganism and throughout its growth.

Inoculum for the preparation of the antibiotic mixture may be obtainedby employing growth from slants of M. Iacustris on such media as ATCCMedium 172 to which previous reference was made. The growth may be usedto inoculate either shake flasks or inoculum tanks, or alternatively,the inoculum tanks may be seeded from the shake flasks. The growth ofthe microorganism usually reaches its maximum in about 2 or 3 days.However, variations in the equipment used, aeration, rate of stirring,and so forth, may affect the speed with which the maximum growth isreached. In general, the fermentation is conducted until substantialantimicrobial activity is imparted to the medium, a period of from about24 hours to about 4 days being sufficient for most purposes.

The process of antibiotic production is conveniently followed duringfermentation by biological assay of the broth employing a sensitivestrain of Staphylococcus aureus. Standard plate assay technique isemployed in which the zone of inhibitation surrounding a filter paperdisc saturated with the broth is used as a measure of antibioticpotency. After the fermentation broth has reached a level of antibioticpotency, the pH is usually about 7.5-8.5, the mycelium is removed byfiltration or centrifugation. Various types of equipment such as filterpresses, centrifuges, etc. may be employed.

Thin layer chromatography employing silica gel is a useful tool foranalyzing the antibiotic mixture produced by M. lacustris infermentation media and the composition of crude and purified materialsextracted from clarified fermentation broths. The resolution of thecomponents of the antibiotic mixture is importantly dependent onantibiotic loading of the system. Too little antibiotic potency fails toreveal minor antibiotic components; too much antibiotic potency resultsin a dragging effect with resulting poor resolution.

The developing system for the thin layer chromatography is the upperlayer prepared from ethyl acetate, tetrahydrofuran and water (4: l :5).Bioautographic detection of the antibiotic compounds may be accomplishedby means of an overlay of a thin layer of agar seeded with a sensitivestrain of Staphylococcus aureus or other sensitive organism. The thinlayer chromatograms, after development, may also be examined visually.The antibiotics present in the antibiotic mixture are all highly coloredwith various shades of orange, yellow and pink.

The rifamycins produced by M. lacustris are compounds of the formulae:

cu 04 H The major portion 90%) of the antibiotic mixture produced by M.lacustris, established by elemental analysis, mass spectrographic andnmr data, is represented by 3-methylthiorifamycin S (llb) and3-thiomethylrifamycin SV (lb). The largest minor components 9%) arerifamycin S (Ila) and rifamycin SV (Ia). Smaller amounts of rifamycin Sand rifamycin SV are produced by the mutant strain M. lacustris ATCC21974.

In addition to the rifamycins, a new ansamycin designated Compound32,656 is produced 0.9%) and a number of other ansamycins in traceamounts totaling about 0.1%. of the antibiotic mixture.

The oxidation-reduction equilibria established for rifamycin S andrifamycin SV exist for the 3-methylthio derivatives of theseantibiotics. Rifamycin SV is oxidized by air or by manganese dioxide torifamycin S. Rifamycin S is readily reduced to rifamycin SV by treatmentwith ascorbic acid. Similar oxidationreduction reactions are operativefor 3-methylthiorifamycin S and 3-methylthiorifamycin SV.

Hydrogenation of 3-methylthiorifamycin S in ethanol solution in thepresence of 5% palladium on carbon until eight equivalents of hydrogenare consumed, removal of catalyst by filtration and evaporation of thefiltrate yields the hexahydro derivative of 3-methylthiorifamycin SV.Oxidation of this compound with activated manganese dioxide gives thehexahydro derivative of 3-methylthiorifamycin S. Similarly, hydrogenation of Compound 32,656 gives the hexahydro deriva tive.

The components of the antibiotic mixture may be recovered fromfermentation broth by a number of different procedures including solventextraction and column chromatography or combinations thereof. Variousorganic solvents are useful in extracting the antibiotics from clarifiedbroth. Methyl isobutyl ketone is a particularly effective solvent.Solvent extract is preferably by adding ascorbic acid to the whole brothbefore filtration at a level of about 2 grams per gram of antibioticmixture and stirring for about 30 minutes at room temperature.Alternatively, the antibiotics may be re- 3 CH3 H S-cH CH O duced withascorbic acid at one of the solvent stages of recovery andconcentration.

The preferred method of separation and recovery of the components of theantibiotic mixture is as follows: The clarified broth is adjusted to pH4.0 to 4.5 and extracted with about l/5 volume of methyl isobutylketone. The ketone is removed under vacuum and replaced with industrialethanol. The ethanol solution is defatted by repeated extraction withpetroleum ether. The ansamycins are reduced with ascorbic acid, theethanol removed under vacuum and the residue taken up in chloroform. Thechloroform is evaporated under vacuum and the residue is chromatographedon a silica gel column using ethyl acetate with increasingconcentrations of acetone as the developing solvent. Column cuts arefollowed by thin layer chromatography and bioassay. The active cuts arecombined accordingly. Compound 32,656 is in the fore cuts along withtrace amounts of other ansamycins. The acetone concentration in theethyl acetate-acetone developing mixture is raised stepwise to about35-50%. The heart out is eluted and concentrated yielding crystalline3-methylthiorifamycin SV.

3-methylthiolrifamycin SV is readily converted to 3-methylthiorifamycinS by oxidation with air or preferably by treatment with activatedmanganese dioxide which is prepared by azeotropic drying of manganesedioxide as described in J. Org. Chem. 34, No. 6, 1979 (1969). A slurryof activated manganese dioxide, approximately a gram per gram ofantibiotic, is added to a methanolic or ethyl acetate solution of theantibiotic and stirred for about 30 minutes at room temperature at whichtime the oxidation is substantially complete. The reaction mixture isclarified by filtration or centrifugation and the solvent removed undervacuum.

The present invention includes within its scope the antibiotic productsproduced by M. lacustris in dilute forms, crude concentrates, and alsothe purified components thereof. All of these novel products are usefulin combatting microorganisms, especially Mycobacterium tuberculosis,Diplocoocus pneumoniae, Streptococcus pyogenes and Staphylococcusaureus, including strains which are resistant to other knownantibiotics. In addition they are useful as disinfectants against suchmicroorganisms and as an aid in the purification of mixed cultures formedical diagnostic and biological research purposes.

Table I illustrates the antibiotic spectra of some of these newansamycins. These tests were run by preparing tubes of nutrient brothwith gradually increasing concentrations of the pure antibiotic and thenseeding the broths with the particular organism specified. The minimalinhibitory concentration indicated in Table l is the minimalconcentration of the antibiotic (in micrograms/milliliter) at which themicroorganism failed to grow. The tests were conducted understandardized conditions as described in Proc. Soc. Exp. Biol. & Med.122, 1107 (1966). Tables II and Ill illustrate the in vivo activity ofthese new antibiotics in experimentally infected mice.

weight polyethylene glycols. When aqueous suspensions and/or elixirs aredesired for oral administration, the essential active ingredient thereinmay be combined with various sweetening or flavoring agents, coloringmatter or dyes, and if desired, emulsifying and/or suspending agents aswell, together with such diluents as water, ethanol, propylene glycol,glycerol and various combinations thereof.

For purposes of parenteral administration, solutions of theseantibiotics in sesame or peanut oil or in aqueous propylene glycol maybe employed as well as sterile aqueous solutions of the correspondingwater-soluble alkali metal or alkaline-earth metal salts. Such aqueousTable I Minimal Inhibitory Concentration g/ml) M. tuber, StreptococcusStaphylococcus S. aureus H Rv pyrogenes aureus (multi-resistant)Antibiotic 3-methylthio- 0.015 0.0019 0.0019 00019-00039 rifamycin SV3-methylthio- 0.006 0.0009 0.0004 0.00l20.0039 rifamycin 8 Compound32,656 0.1 0.03l2 0.0039 0.00l20.0078

Table II P mg/kg' Diplucucci Streptococci Staphylococci Antibiotic oraloral oral subcutaneous B-methylthio- 8.0 0.22 1.1 0.65 rifamycin SV3-methylthiol2.5 0.60 1.4 0.17 rifamycin S Compound 32,656 l.l

" Dnse that provides 50% protection Table III solutions should besuitably buffered if necessary and M. tuberculosis Infection in Mice*Evaluatcd at 53 days post challenge The antibiotics of these inventioncan be administered via the oral or parenteral routes for the treatmentin animals, including humans, of pneumococcal, streptococcal,staphylococcal, tubercular and other antibiotic-sensitive infections. Ingeneral, these antibiotics are most desirably administered in daily oraldoses of 0.5-1 gram or parenteral injections of 100 to 500 mg.,depending on the type and severity of the infection and weight of thesubject being treated.

The compounds of this invention may be administered alone or incombination with pharmaceuticallyacceptable cariers, and suchadministration can be carried out in both single and multiple doses.

For purposes of oral administration, tablet containing variousexcipients such as sodium citrate, calcium carbonate and dicalciumphosphate may be employed along with various disintegrants such asstarch, alginic I the liquid diluent first rendered isotonic withsufficient saline or glucose.

The following examples are given to more fully illustrate the invention.It is to be understood that these examples are for illustrative purposesonly and that the invention is not meant to be limited to the specificdetails of the examples.

EXAMPLE I A sterile aqueous medium having the following composition isprepared:

Grams/liter Starch 20.0 Enzymatic digest of casein 5.0 Yeast extract 5.0Dextrose 10.0 K HPO 0.5 CaCO 4.0

Cells from a slant of M. lacustris ATCC 21975 are transferred to aseries of 3-liter Fernbach flasks each containing a liter of this mediumand shaken for 3-4 days at 28C.

About 1% v/v of the grown inoculum is transferred to a fermentercontaining gallons of sterile medium of the following composition:

Grams/liter Starch 25.0 Enzymatic digest of casein dl-MethionineAmmonium acetate Ammonium sulfate K HPO,

Sucrose CaCO Meat meal CoCl .6H O

The temperature is maintained at about 30C. and the broth is stirred at1 150 rpm and aerated at the rate of /2 volume of air per volume ofbroth per minute. After 35-45 hours, 5% v/v of this grown inoculum istransferred to a fermenter containing 1000 gallons of the sterile mediumdescribed above. The temperature is maintained at 30C., aeration at /2volume of air per volume of broth per minute and stirring at 600 rpm.Soybean oil is added as needed to control excessive foaming. After 5060hours, 5l0% v/v of this grown inoculum is transferred to a fermentercontaining 10,000 gallons of sterile medium which is the same as thatdescribed above but without meat meal. The temperature is maintained at30C., aeration at the rate of /2 volume of air per volume of broth perminute and stirring at 380 rpm. Soybean oil is added as necessary tocontrol foam. Sucrose or dextrose is added at 24 hour intervals to alevel of about 0.1% w/v.

After 90120 hours, the fermentation broth is fil tered and the pHadjusted to 4.0-4.5 in the presence of 5-10% methyl isobutyl ketone. Thebroth is then extracted with 2500 gallons of methyl isobutyl ketone bymeans of a Pokbielniak extractor and centrifugal separator. The solventextract is concentrated under vacuum to l/-1/20 volume and the pHadjusted to 5.5-6.5 with ammonia or 10% K HPO solution.

The solvent (approximately 200 gallons) is removed under vacuum andreplaced with 3A ethanol. The ethanol solution is concentrated undervacuum to about gallons which is defatted by repeated extraction withpetroleum ether. To the ethanolic solution is added ascorbic acid (about2 grams/gram of antibiotic mixture) and the solution is stirred at roomtemperature for about 30 minutes. The ethanolic solution is concentratedunder vacuum, maintaining the pH at 6-7 if necessary, to a thin syrupwhich is then taken up in chloroform.

The chloroform solution is concentrated to a syrup which is thenchromatographed on a silica gel column and developed with ethyl acetatecontaining increasing 5% increments of acetone. The column cuts arefollowed by thin layer chromatography and bioassay, and the cuts arecombined accordingly. The fore cuts contain Compound 32,656 and traceamounts of rifamycin S, rifamycin SV and other ansamycins. The heart cuteluate when concentrated yields crystalline 3-methylthiolrifamycin SV.

EXAMPLE II The method of Example I is repeated with M. lacustris ATCC21974 in place of ATCC 21975. Comparable results are obtained except forthe fermentation production of smaller amounts of rifamycin S andrifamycin SV.

EXAMPLE III A dry solid pharmaceutical composition is prepared byblending the following maerials together in the proportions by weightspecified below:

3-Methylthiorifamycin SV 50 Sodium citrate Alginic acid 10Polyvinylpyrrolidone 10 Magnesium stearate 5 After the dried compositionis thoroughly blended, tablets are punched from the resulting mixture,each tablet being of such size that it contains 500 mg. of the activeingredient. Other tablets are prepared in a similar manner containing250, and 50 mg. of the active ingredient by using the appropriate amountof 3-methylthiorifamycin SV in each case.

EXAMPLE IV Vials are prepared containing weighed amounts of the sterilesodium salt of 3-methylthiorifanycin SV. These vials are reconstitutedfor parenteral administration to 100 or 200 mg/ml with sterile water or5% dextrose solution.

EXAMPLE V Analytical Data 3-Methylthiorifamycin S Carbon 61.48 Hydrogen6.70 Nitrogen l .90 Sulfur 4. l 5

Mass spectrometer and nmr data are consistent with 3-thiomethy1rifamycinS (C H O NS). lts ultraviolet absorption maxima in 0.01 M l-lCl inmethanol solution occur at 220, 265, 309 and 373 m,u..

A KBr pellet shows characteristic absorption in the infrared region atthe following wavelengths in microns: 2.90, 3.40, 5.70, 5.80, 5.95,6.15, 6.35, 6.80, 7.05, 7.25, 7.60, 7.75, 8.00, 8.40, 8.55, 8.90, 9.30,9.50, 10.30, 10.60. 10.80, 11.20 and 12.00.

3-Methylthiorifamcyin SV (sodium salt) Carbon 56.24 Hydrogen 6.47Nitrogen l .59 Sulfur 3 .50

Rifamycin S The identity of rifamycin S produced by M. lacustris wasestablished by elementary analysis and comparisons with publishedinfrared and ultraviolet light absorption spectra.

Rifamycin SV The identity of rifamycin SV produced by M. lacustris wasestablished by elementary analysis and comparisons of infrared andultraviolet light absorption spectra with published data.

Compound 32,656

Carbon 58. I97: Hydrogen 5.97% Nitrogen 3. l 7% Sulfur 3.52% Oxygen (bydifference) 29.157:

EXAMPLE VI Hydrogenation of an ethanolic solution of3-methylthiorifamycin S in the presence of 5% palladium on carbon until8 equivalents of hydrogen are consumed,'removal of the catalyst byfiltration and evaporation of the filtrate yielded the hexahydroderivative of 3-thiomethylrifamycin SV, identified by mass spectrometerdata. Oxidation of this compound with activated manganese dioxide yieldshexahydro-3-methylthiorifamycin S. The antibiotic properties of thesehexahydro derivatives are comparable to those of the parent compounds.

EXAMPLE VII The hexahydro derivative of Compound 32,65 6, withantibiotic activity comparable to that of the parent compound, isprepared by hydrogenating Compound 32,656 by the method of Example VI.

What is claimed is:

l. 3-Methylthiorifamycin S.

2. 3-Methylthiorifamycin SV.

3. The hexahydro derivative of 3-methylthiorifamycin S.

4. The hexahydro derivative of 3-methylthiorifamycin SV.

UNITED STATES PATENT AND TRADEMARK OFFICE CERTIFICATE OF CORRECTIONvPATENT N0. 3 914, 218

DATED 3 October 21, 1975 |NVENTOR(S) Celmer et al It is certified thaterror appears in the above-identified patent and that said LettersPatent are hereby corrected as shown below:

In the Abstract change "3-thiomethyl" to 3-methylthio after "328.

Col. 2 line 7/insert But with 3g glucose in place of sucrose and noagar.

Col. 2 line 34 change "These" to there Col. 5 line 26-27 change"3-thiomethyl" to 3m thy1thi Cole 6 under the heading "3" change (a) K"to (a) H C010 7 line 46 change "invention" to inventions Col. 9 line 23change "Pokbielniak" to Podbielniak.-.

Col. 9 line 47 change 3methylthiolrifamycin" to 3-methylthiorifamycinline Col. 10/23 change "3-thiomethyl" to 3methylthio Col 11 line 20change "3-thiomethyl" to 3-methylthio ,Erigncd and Scaled this Tenth DayOf August 1976 [SEAL] Arrest:

RUTH C. MASON C. MARSHALL DANN Arresting Officer Commissioner nfPatentsand Trademarks

1. 3-METHYLTHIORIFAMYCIN S.
 2. 3-Methylthiorifamycin SV.
 3. Thehexahydro derivative of 3-methylthiorifamycin S.
 4. The hexahydroderivative of 3-methylthiorifamycin SV.